The goal of this research is to understand the molecular mechanisms underlying quorum sensing, a cell-cell communication process that allows bacteria to coordinate population-wide gene expression and function as coordinated groups. Quorum sensing relies on the production, detection, and response to chemical signal molecules called autoinducers. Disruption of any one of these steps renders pathogenic bacteria avirulent. The research proposed here will provide an understanding of the molecular mechanisms underpinning signal production, detection and response in quorum-sensing which will enable the design of interference strategies that can be developed into new therapies to combat infectious diseases. The human pathogen Vibrio cholerae, the causative agent of the disease cholera, regulates virulence factor production and biofilm formation through quorum sensing via a new class of autoinducer, called CAI-1 ((S)-3-hydroxytridecan-4-one). CAI-1 is produced by the CqsA synthase and detected by the transmembrane histidine kinase receptor CqsS. Histidine kinase receptors are ubiquitous in bacteria and play important roles in bacterial pathogenesis, nonetheless, little is known about their molecular mechanism of signal transduction across the membrane. This is, in part, due to a profound lack of chemically defined ligands for histidine kinases. By determining the biosynthetic pathway for and structure of CAI-1, and examining the CAI-1-CqsS interaction as well interactions between CqsS and a series of related, synthetic molecules, my postdoctoral work demonstrated that the CqsA/CqsS/CAI-1 signaling pathway offers an unprecedented opportunity to study ligand-receptor interactions and transmembrane signaling by histidine kinase receptors. With this work as the foundation, going forward, my first aim is to use CAI-1/CqsS as a model system to define how this important class of receptors recognizes and propagates external signals internally into the cell. My second aim focuses on co-evolution of signal production by the CqsA synthases and signal recognition by the partner CqsS receptors. As a postdoc, I showed that signal production and signal recognition in each vibrio CqsA/CqsS pair are matched. That is, either a particular CqsA synthesizes multiple related molecules and the cognate CqsS receptor detects them all, or the CqsA enzyme produces one specific molecule and the cognate CqsS receptor is exquisitely selective for detection of that ligand. The biological significance of this co-evolution and the molecular mechanism that specifies stringency or lack thereof are unclear and I will study this phenomenon. Our understanding of the role quorum sensing plays in V. cholerae physiology is limited to virulence factor production and biofilm formation. However, the V. cholerae quorum-sensing regulon is composed of more than 100 genes in addition to those known to be involved in virulence and biofilm formation. My recent work showed that V. cholerae mutants unable to activate a quorum-sensing response are exquisitely sensitive to low pH. In my final aim, I will study the molecular mechanisms underpinning quorum-sensing dependent regulation of Acid Tolerance Response. The long term goal is to define the roles of V. cholerae quorum-sensing response in coping with the changes in the environment and in the host. It is now well established that quorum sensing is employed by many bacterial species to regulate both harmful and beneficial traits. A long standing goal of the quorum-sensing field is to develop pro-quorum- sensing and/or anti-quorum-sensing small molecules to manipulate these behaviors. This study will make progress toward this important goal. PUBLIC HELATH RELEVANCE: Vibrio cholerae is a globally important pathogen and the burden of cholera is estimated to reach several million cases annually. V. cholerae virulence depends on a cell-cell communication process called quorum sensing that controls the timing of production and release of virulence factors and the formation of biofilms. The investigations proposed here will expand our understanding of how quorum sensing controls virulence in this important pathogen, in addition, this study will facilitate the development of synthetic strategies for controlling V. cholerae virulence and could have enormous ramifications for improving human health.